缺氧/放射活化Epo/e9增强子调控layilinsiRNA表达以抑制人肺腺癌A549细胞侵袭的研究

摘要

背景与目的:Layilin是一种新发现与肺腺癌侵袭转移有密切联系的特异性透明质酸受体.本研究旨在观察缺氧/放射条件下活化缺氧/放射双敏感性增强子(Epo/e9),调控针对layilin的小干扰RNA (small interfering RNA,siRNA)表达,探讨其对人肺腺痛A549细胞受透明质酸诱导侵袭的影响.方法:构建携带Epo/e9增强子和U6启动子且表达针对layilin的siRNA的RNA干扰载体(Epo/e9-siLay plasmid)和阴性对照RNA干扰载体(Epo/e9-siCtrl plasmid),分别转染A549细胞,新霉素抗性筛选得到layilin表达受抑制的A549Epo/e9-siLay细胞和layilin表达未受影响的A549Epo/e9-siCtrl细胞.针对非转染A549(A549untransfected)、A549EPo/e9-SiLay、A549Epo/e9-siCtrl三组细胞,在缺氧/放射处理下,分别采用RT-PCR和Western blot检测layilin mRNA和蛋白表达,用Transwell模型检测细胞侵袭能力.结果:与A549untransfected组相比,A549Epo/e9-siLay组layilin表达显著减弱(P＜0.01),Transwell穿膜细胞数显著减少(P＜0.01)；A549Epo/e9-siCtrl组layilin表达、Transwell穿膜细胞数皆和A549untransfected组无显著差异(P＞0.05).缺氧/放射处理能进一步增加RNA干扰的上述效应(P＜0.01).结论:在缺氧/放射条件下,Epo/e9增强子调控layilin siRNA表达能明显抑制A549细胞侵袭行为.%Background and purpose: Layilin is a novel and special hyaluronan (HA) receptor that closely linked with lung cancer invasion and metastasis. The objective of this study is to investigate the effect of hypoxia/ radiation dual-sensitive enhancer (Epo/e9) regulating small interfering RNA (siRNA) for layilin expression on invasion of human lung adenocarcinoma A549 cells induced by HA in vitro. Methods: RNA interference plasmid including Epo/e9 enhancer and U6 promoter and expressing siRNA targeting layilin (Epo/e9-siLay plasmid) or expressing siRNA not matching any known human coding Mrna (Epo/e9siCtrl plasmid) was designed, constructed, and lipotransfected into A549 cells line. A549Epo/e9-siLay cells expressing suppressed layilin or A549Epo/e9-siCtrl cells expressing uninfluenced layilin were selected by neomycin resistance. In A549untransfected, A549Epo/e9-siLay and A549Epo/e9-siClrl groups, layilin Mrna and protein expression were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, and the invasion ability was examined by Transwell model with or without administration of hypoxia/radiation. Results: Compared with that in A549untransfected group, layilin Mrna and protein expression, and the number of penetrating cells (NPC) were decreased significantly in A549Epo/e9-siLay group (P<0.01), but there were no significant changes in A549Epo/e9-sictri group (P>0.05). Administration of hypoxia/radiation reinforced the above effects of siRNA targeting layilin. Conclusion: Epo/e9 enhancer regulating siRNA for layilin expression can inhibit efficiently the invasive ability of human lung carcinoma cell line A549 induced by HA in vitro with hypoxia/radiation administration.